Cardiac output adjusts on a beat-to-beat basis due to the changing balance of parasympathetic and sympathetic input to the heart. G protein signaling pathways are fundamental to this important process. Indeed, the prototypical signaling pathway consisting of the type 2 muscarinic acetylcholine receptor (m2R) and the G protein-gated atrial potassium channel IKACh mediates in large part the inhibitory effects of parasympathetic activity on the heart. Too much or too little parasympathetic influence on the heart can trigger arrhythmias, often with fatal consequences. The long-term goal of our research is to identify and characterize molecular mechanisms that control or impact m2R-IKACh signaling, and consequently, the parasympathetic regulation of the heart. This application capitalizes on the recent discovery that IKACh associates physically with a regulatory complex consisting of the sixth member of the Regulator of G protein Signaling protein family (Rgs6) and the fifth member of the G protein beta subunit family (G5). The interaction between Rgs6/ G5 and IKACh has clear functional implications, as temporal aspects of m2R-IKACh signaling are altered in atrial myocytes from mice lacking Rgs6. Together, these preliminary data suggest the central hypothesis of this proposal, namely that Rgs6/ G5 serves as a negative regulator of m2R-IKACh signaling in the heart, with a corresponding and predictable influence on the parasympathetic control of cardiac output. This hypothesis will be tested by pursuing three complementary Specific Aims: (1) To understand the molecular organization and functional significance of the Rgs6/ G5 -IKACh complex, (2) To define the impact of Rgs6/ G5 on m2R-IKACh signaling in atrial myocytes, and (3) To understand the significance of the Rgs6/ G5 complex to cardiac physiology. The strategy proposed to address these aims will entail a synergistic combination of biochemical, molecular biological, electrophysiological, and physiological approaches, each exploiting the existence of a powerful array of reagents and animal models. Successful completion of these studies will yield detailed insights into the molecular determinants of Rgs6/ G5 -IKACh complex assembly and a clear understanding of the functional correlates of complex formation on m2R-IKACh signaling as assessed in the well-controlled expression system, the native context of the atrial myocyte, and the whole animal. By leveraging the unique strengths and research infrastructures of two laboratories, this project is poised to reveal novel insights into the organization and regulation of G protein signaling pathways and the parasympathetic regulation of cardiac output. As such, this information could prove useful for the development of novel therapeutic interventions designed to prevent or treat certain types of arrhythmia. PUBLIC HEALTH RELEVANCE: Normal heart function depends on a healthy balance between excitatory and inhibitory signals provided by the nervous system. Imbalance in this critical cardiac regulatory system can trigger arrhythmias, often with fatal consequences. The work proposed herein will yield a clearer understanding of the molecules involved in the regulation of cardiac output by the nervous system and thus will aid in the rational design of effective therapeutic interventions aimed at treating or preventing arrhythmias.